Abstract, Project 3 A premise of current research on vaccines for rapidly evolving pathogens (e.g., influenza virus and HIV) is that better understanding of molecular events during antibody affinity maturation can lead to more rational vaccine design. The goals of Project 3 are to interpret, in biophysical and structural terms, the findings from immunization studies with humans, non-human primates, and mice in Projects 1 and 2; to define, with quantitative experimental results, concepts such as immunodominance and epitopic distance; to provide molecular movies of specific affinity maturation trajectories; and to design new immunogens, as tests for the hypotheses explored and generated in the Program Project as a whole. We concentrate on antibodies that target the receptor-binding site (RBS). (1) We will generate a structural epitope map of the nave response to a single HA subtype, to provide an epitope language for the rest of the project. We will then seek structural explanations for any heterosubtypic antibodies in the nave response (human, macaque) to a multivalent vaccine. (2) Can we use a set of HAs with defined similarity at a particular epitope (epitopic distance) to determine the extent to which a primary exposure will dominate a subsequent, heterologous humoral response? We propose an operational definition of epitopic distance and outline experiments to test and modify it. We will relate epitopic distance to structural features, with the long-term goal of enabling computational estimates from knowledge of a single-Fab:single-HA-variant structure. This approach to the HA RBS, an extremely well characterized epitope, will establish benchmarks and concepts to apply more broadly to design of immunogens and vaccination regimens. (3) Building on (1) and (2), we will test directly and critically the concept of B-cell lineage-based immunogen design, while also testing the outcomes, in mice and macaques (Projects 2 and 1, respectively), of particular immunization protocols with particular designed immunogens.